GSM7049196: antiH3 ChIP in nrpe1 rep2; Arabidopsis thaliana; ChIP-Seq
All the material for ChIP used in this paper were from pooled 10-12 day old seedlings grown on Murashinge and Skoog agar plates (1/2X MS, 1.5% agar, pH5.7). 2-4g of seedling tissue was used for each sample. Samples were ground in liquid nitrogen and crosslinked for 10 or 12 minutes at room temperature in Nuclei Isolation buffer (50mM Hepes pH8, 1M sucrose, 5mM KCl, 5mM MgCl2, 0.6% triton X-100, 0.4 mM PMSF, 5 mM benzamidine hydrochloride, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)) containing 1% formaldehyde. The crosslinking reactions were stopped with 125 mM glycine by incubation at room temperature for 10 minutes. Crosslinked nuclei were filtered through one layer of miracloth, washed with Extraction Buffer 2, centrifuged through a layer of Extraction Buffer 3 and lysed with Nuclei lysis buffer (buffer compositions are described in the published protocol (Villar and Kohler, 2018)). Chromatin was sheared using a Bioruptor Plus (Diagenode) (20 cycles of 30 seconds on and 30 seconds off) and immunoprecipitated overnight at 4°C with antibody. anti-NRPE1 (endogenous antibody, described in (Liu et al., 2018)), anti-H1 (AS11 1801 Agrisera), anti-H3K9me2 (abcam1220), anti-H3 (abcam1791), anti-FLAG (for both the SUVH1-3xFLAG and the DMS3-ZF ChIPs, we used Anti-FLAG M2 (Sigma, F1804)). 25 µl each of Protein A and Protein G magnetic Dynabeads (Invitrogen) were added to each sample and incubated for 2 more hours at 4°C. The immunoprecipitated chromatin was washed with Low Salt (2X), High Salt, LiCl and TE buffers, for 5 minutes each at 4°C (buffer compositions are described in the published protocol (Villar and Kohler, 2018)) and eluted twice with 250 µl of elution buffer (1% SDS and 0.1 M NaHCO3) in a thermomixer at 65°C and 1000 rpm, 20 minutes for each elution. Reverse crosslinking was performed overnight at 65°C in 0.2 M NaCl, and proteins were degraded by proteinase K treatment at 45°C for 5h. The DNA fragments were purified using phenol:chloroform and ethanol precipitated overnight at -20°C. Libraries were prepared using the Ovation Ultra Low System V2 1-16 kit (NuGEN, 0344NB-A01) following the manufacturer's instructions, with 15 cycles of PCR. Libraries were sequences on a Novaseq 6000 using either single- or paired-end 50bp reads. Whole genome bisulfite sequencing libraries were generated as previously described (Harris et al., 2016), with minor modifications. The DNeasy Plant Mini Kit (Qiagen #69104) was used to isolate genomic DNA. 150 ug of genomic DNA was used as input, a Covais S2 instrument was used for shearing (2 minutes), the Epitect Kit (QIAgen #59104) was used for bisulfite conversion, and the Nugen Ultralow Methyl Kit was used for library preparation and paired-end libraries were sequences on a Novaseq 6000 instrument.